SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Determination of the orientation of T4 lysozyme
vectorially bound to a planar supported lipid bilayer
using site-directed spin labeling
Kerstin Jacobsen 1, Shirley Oga 2, Wayne L. Hubbell 2 and Thomas Risse 3*
1 Fritz-Haber Institute
2 Jules Stein Eye Institute
3 Fritz-Haber-Institute
* To whom correspondence should be addressed. E-mail: risse{at}fhi-berlin.mpg.de.
Submitted on January 21, 2005
Revised on March 2, 2005
Accepted on 14 March 2005
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Abstract |
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Site-directed spin labeling is used to investigate the structure of adsorbed T4 lysozyme (T4L). A monolayer of T4L is prepared by tethering the protein selectively via a His-tag to the chelating headgroups (NTA Ni) of a planar quartz-supported lipid bilayer. This results in a vectorially oriented ensemble of proteins on the surface, which gives rise to angular dependent EPR spectra. Similar measurements of spin labeled lipid bilayers were used to characterize the structure and dynamics of the supports. EPR line shape was analyzed using the stochastic Liouville equation (SLE) approach developed by Freed and coworkers. The simulations reveal a conservation of the secondary and tertiary structure of T4L upon adsorption although slight conformational changes in the presence of the surface can be detected by probing tertiary contact sites. The orientation of the entire protein was deduced on the basis of an anisotropic motional model for the spin labeled side chain. In addition, a polar order but azimuthal disorder of the molecules was assumed to fit the data. These results demonstrate the utility of SDSL in combination with spectral simulation to study not only the secondary and tertiary structure of adsorbed proteins in monolayer coverage but also their orientation with respect to the surface.
Key Words:
EPR spectroscopy, Side chain dynamics, T4 Lysozyme, lipid-protein interaction, site directed spin labeling, supported lipid bilayer
